Differential distribution of eicosanoids and polyunsaturated fatty acids in the Penaeus monodon male reproductive tract and their effects on total sperm counts

Eicosanoids, which are oxygenated derivatives of polyunsaturated fatty acids (PUFAs), serve as signaling molecules that regulate spermatogenesis in mammals. However, their roles in crustacean sperm development remain unknown. In this study, the testis and vas deferens of the black tiger shrimp Penaeus monodon were analyzed using ultra-high performance liquid chromatography coupled with Orbitrap high resolution mass spectrometry. This led to the identification of three PUFAs and ten eicosanoids, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and (±)15-hydroxyeicosapentaenoic acid ((±)15-HEPE), both of which have not previously been reported in crustaceans. The comparison between wild-caught and domesticated shrimp revealed that wild-caught shrimp had higher sperm counts, higher levels of (±)8-HEPE in testes, and higher levels of prostaglandin E2 (PGE2) and prostaglandin F2α in vas deferens than domesticated shrimp. In contrast, domesticated shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and eicosapentaenoic acid (EPA) in testes and higher levels of 15d-PGJ2, (±)12-HEPE, EPA, arachidonic acid (ARA), and docosahexaenoic acid (DHA) in vas deferens than wild-caught shrimp. To improve total sperm counts in domesticated shrimp, these broodstocks were fed with polychaetes, which contained higher levels of PUFAs than commercial feed pellets. Polychaete-fed shrimp produced higher total sperm counts and higher levels of PGE2 in vas deferens than pellet-fed shrimp. In contrast, pellet-fed shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and EPA in testes and higher levels of (±)12-HEPE in vas deferens than polychaete-fed shrimp. These data suggest a positive correlation between high levels of PGE2 in vas deferens and high total sperm counts as well as a negative correlation between (±)12-HEPE in both shrimp testis and vas deferens and total sperm counts. Our analysis not only confirms the presence of PUFAs and eicosanoids in crustacean male reproductive organs, but also suggests that the eicosanoid biosynthesis pathway may serve as a potential target to improve sperm production in shrimp.

There has been limited information regarding the roles of eicosanoids in crustacean sperm development. A study in wild Litopenaeus occidentalis revealed that the administration of ibuprofen, which inhibits prostaglandin biosynthesis, increased normal spermatophore development [16]. This suggests a negative correlation between prostaglandin biosynthesis pathway and spermatogenesis in shrimp. On the other hand, high levels of dietary polyunsaturated fatty acids (PUFAs) showed a positive impact on crustacean sperm production [17,18].
To further explore the roles of eicosanoids and PUFAs in crustacean spermatogenesis, P. monodon testes and vas deferens were subjected to liquid-liquid extraction and ultra-high performance liquid chromatography coupled with Orbitrap high resolution mass spectrometry (UHPLC-HRMS/MS) analysis. Levels of eicosanoids and PUFAs in testes and vas deferens were then compared between those of wild-caught and domesticated shrimp, which had high and low sperm counts, respectively. The effects of shrimp feed on eicosanoid and PUFA profiles in testes and vas deferens of domesticated shrimp were also examined. Our findings confirm the presence of eicosanoids in shrimp male reproductive tract and suggest that the roles of eicosanoids in regulating total sperm number in crustaceans are conserved relative to mammals.

Ethical statement
All experiments were approved by the Institutional Animal Care and Use Committee of the National Center for Genetic Engineering and Biotechnology, Thailand (Approval Code BT-Animal 13/2560). This permit covered the purchase wild-caught shrimp, shrimp transportation, shrimp rearing experiment, and shrimp dissection. No permit was required for the collection site access as the wild-caught broodstock collection from the Andaman Sea was conducted by local fishermen and purchased through a local shrimp farm. All experiments were performed in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) and conformed with international and national legal and ethical requirements, including the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, and the National Research Council's Guide for the Care and Use of Laboratory Animals.

Shrimp sources
Wild-caught male shrimp were captured from the Andaman Sea, Thailand (salinity level at approximately 31 ppm) (N = 10). Eleven-month-old domesticated male P. monodon, which had been raised in earthen ponds and fed with commercial feed pellets, were acquired from the Shrimp Genetic Improvement Center (SGIC), Surat Thani, Thailand (N = 10). Average body weights of wild-caught and domesticated shrimp were 86.9 ± 9.0 and 66.8 ± 7.6 g, respectively. Shrimp testes and vas deferens were dissected and flash frozen in liquid N 2 for the quantification of eicosanoids and PUFAs using UHPLC-HRMS/MS. Spermatophores were collected and used for total sperm counts.

Effects of shrimp feed
To determine changes in eicosanoid and PUFA levels in shrimp fed with different diets, elevenmonth-old, domesticated males from the SGIC were fed with either polychaetes or feed pellets for four weeks (N = 8 each). Fatty acid profiles in polychaetes and feed pellets (N = 4 per feed) were analyzed using gas chromatography coupled with flame ionization detector (GC-FID) by the Nutrition Service at Central Lab Co., Ltd. (Thailand) (www.centrallabthai.com). Shrimp testes and vas deferens were dissected and flash frozen in liquid N 2 . Spermatophores were collected and used to determine total sperm counts and percentage of sperm abnormality.

Total sperm counts and sperm abnormality
Spermatophores were individually homogenized in a calcium-free sea water solution. After debris sedimentation, sperms were counted using a hemocytometer under a light microscope [19]. Abnormal sperms were defined as sperms with a misshaped head or tail as well as sperms with no head or tail [20]. Total sperm counts and abnormal sperm counts were determined from both spermatophores of each shrimp using average counts of four aliquots from each spermatophore homogenate. The percentage of abnormal sperm were then calculated based the percentage of abnormal sperm from the number of total live sperm.

Sample preparation
Shrimp testes and vas deferens were individually homogenized in liquid N 2 and diluted in HBSS to adjust tissue concentration to 0.1 g/mL (wet weight). Organ homogenates were divided into 500 μL aliquots and adjusted to pH 4.0 using 5 μL of glacial acetic acid. Ten microliters of 10% BHT in HPLC-grade ethanol (w/v) were added as an antioxidant. Internal standards, including PGE 2 -d 4 , 5(S)-HETE-d 8 , and EPA-d 5 , were added to determine the percent recovery in each sample. An optimal extraction method was selected for each organ based on the recovery yields of the internal standards (S1 Table).

Ethyl acetate extraction
Five hundred microliters of testis homogenates were subjected to ethyl acetate extraction at a 1:1 ratio (v/v) of tissue homogenate to ethyl acetate. Extraction mixtures were shaken in the dark for 15 min at 290 rpm and spun down at 8,000 rpm (8,228 ×g) for 10 min at 20˚C. The organic phase (upper phase) was collected, and the extraction process was repeated one more time. The extracts were evaporated to dryness and dissolved with 200 μL of 100% HPLC-grade ethanol for UHPLC-HRMS/MS analysis.

Methanol-chloroform extraction
Five hundred microliters of vas deferens homogenates were subjected to methanol-chloroform extraction using the procedure modified from Folch extraction method [21]. Tissue homogenates were sequentially mixed with 3.75 mL of methanol, 6.25 mL of chloroform, and 3.12 mL of water. Samples were mixed rigorously for 1 min after each solvent was added. The mixture was shaken for 15 min at 290 rpm at room temperature and spun down at 8,000 rpm at 20˚C for 10 min. The organic phase (lower phase) was collected in a clean tube. The extraction was repeated by adding 3.75 mL of chloroform to the remaining aqueous phase. The mixture was vortexed for 1 min, shaken for 15 min at 290 rpm, and then spun down at 8,000 rpm at 20˚C for 10 min. The organic phase was collected, pooled, dried, and dissolved with 200 μL of 100% HPLC-grade ethanol for UHPLC-HRMS/MS analysis.

UHPLC-HRMS/MS analysis
Chromatographic separation was performed on a Dionex UltiMate 3000RS UHPLC system (Thermo Fisher Scientific) with an Acclaim TM RSLC 120 C18 column (2.1×150 mm, 2.2 μm; Thermo Fisher Scientific) under gradient conditions using mobile phase A (0.01% (v/v) acetic acid in water) and B (0.01% (v/v) acetic acid in acetonitrile) as previously described [22]. The linear gradient went from 30% B to 100% B within 17 min, followed by holding 100% B for 2 min. The elution gradient was returned to the starting condition of 30% B within 0.5 min and kept constant for 3.5 min before starting the next injection. UHPLC conditions included setting auto-sampler temperature at 10˚C, column temperature at 40˚C, injection volume at 5 μL, and flow rate at 300 μL/min for a total run time of 23 min.
Mass spectrometry analyses were performed on an Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Thermo Scientific), equipped with electrospray ionization (ESI) source, and operated in negative ion mode. The mass spectrometer was controlled by the Xcalibur software (version 4.4.16.14) and calibrated using the ESI negative ion calibration solution (Pierce1 LTQ velos ESI negative ion calibration) according to the manufacturer's protocol. Conditions for the mass spectrometer were set with the ESI voltage at 2,500 V in negative mode. Nitrogen was used as the sheath gas at 40 psi and as the auxiliary gas at 12 psi. Ultra-pure helium was used as the collision gas with the ion transfer tube temperature at 333˚C. The vaporizer temperature was 317˚C. Fragment ions of PUFA and eicosanoid standards were detected by the Orbitrap analyzer operated under target mass resolution of 120,000 with an automatic gain control (AGC) setting of 5×10 4 and a maximum ion injection time of 250 ms. The time-scheduled parallel reaction monitoring (PRM) method was used for data acquisition. Analytical characteristics of PUFA and eicosanoid standards used to identify and quantify the compounds in P. monodon tissues are provided in S2 Table. Both limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation (SD) of the response as well as the slope [23,24].

Statistical analysis
Significant differences between the means of independent samples from the two sets of samples were assessed using the t-test with the threshold for significance set at P < 0.05 ( � , † and #) or P < 0.01 ( �� , † † and ##).

Comparison between wild-caught and domesticated males
Wild-caught male P. monodon broodstocks were captured from the Andaman Sea, Thailand ( Fig 1A). Shrimp body weight and body length were recorded prior to dissection to obtain testes, vas deferens, and spermatophores (Fig 1B-1D). Similarly, domesticated males were obtained from SGIC, a biosecure facility located in Surat Thani Province, Thailand ( Fig 1E). Their testes, vas deferens, and spermatophores were also collected ( Fig 1F-1H). It should be noted that all shrimp spermatophores were intact without melanization. Data analysis revealed that wild-caught shrimp had larger body weight (Fig 1I), longer body length (Fig 1J), and higher spermatophore weight ( Fig 1K) than those of domesticated shrimp. Additionally, the total sperm counts of wild-caught shrimp were also higher than those of domesticated shrimp ( Fig 1L).

Heat map visualization of eicosanoids and PUFAs in testes and vas deferens of wild-caught and domesticated shrimp
Heat map analysis was used to compare relative levels of eicosanoids and PUFAs based on the AUC ratio obtained from the UHPLC-HRMS/MS analysis (Fig 4). Testes of wild-caught shrimp contained seven eicosanoids, including PGE 2 , PGF 2α , 15d-PGJ 2 , (±)8-HETE, 12(R)- HETE, (±)8-HEPE, and (±)12-HEPE (Fig 4A, upper panel). Among these, PGF 2α , (±)8-HETE, and (±)8-HEPE were present with high intensities in the heat map, suggesting that these eicosanoids may play crucial roles in spermatogenesis. Additionally, all three PUFAs were present in shrimp testes, in which ARA, DHA, and EPA were detected at low, medium, and high intensities relative to one another, respectively.
UHPLC-HRMS/MS analysis revealed that eight eicosanoids and three PUFAs were detected in vas deferens of wild-caught shrimp. In addition to the seven eicosanoids previously identified in testes, (±)18-HEPE was present in vas deferens with low intensities in the heat map ( Fig 4A, lower panel). In contrast, (±)8-HETE and (±)8-HEPE were present with high intensities in vas deferens. Relative levels of ARA, EPA, and DHA in vas deferens were also similar to those in testes of wild-caught shrimp.
Heat map analysis of eicosanoids and PUFAs in testes and vas deferens of domesticated shrimp revealed different patterns from those in wild-caught shrimp. Three PUFAs and ten eicosanoids were detected in both testes and vas deferens of domesticated shrimp. The two additional eicosanoids identified only in domesticated shrimp were (±)11-HETE and (±) 15-HEPE, which were detected at low intensities in both testes and vas deferens. When relative levels of eicosanoids were examined in testes of domesticated shrimp, it was observed that all ten eicosanoids were present at relatively low intensities in the heat map, which was different from the pattern observed in testes of wild-caught shrimp. On the other hand, the heat map of vas deferens of domesticated shrimp displayed similar metabolic profiles to those of wildcaught shrimp, in which (±)8-HETE and (±)8-HEPE were major products of this pathway.  Metabolite intensities are displayed as colors ranging from yellow to black as shown in the color bar. White indicates that the metabolite was not detected.
Moreover, EPA was consistently the most abundant metabolite in testes and vas deferens of both wild-caught and domesticated shrimp, which illustrates the importance of EPA in the P. monodon sperm maturation process.

Effects of shrimp feed on eicosanoids and PUFAs in the male reproductive tract
In hatcheries, domesticated males are typically fed with live Perinereis nuntia polychaetes instead of commercial feed pellets to increase total sperm counts. To test the effects of shrimp feed on PUFA and eicosanoid profiles in male reproductive tract, eleven-month-old, domesticated males from the same genetic background were fed with either polychaetes or feed pellets for four weeks. Polychaetes and feed pellets were analyzed using GC-FID, revealing that polychaetes contained higher levels of total saturated fatty acids, monounsaturated fatty acids, and  N = 10). Data are shown as means ± SD. Asterisks indicate statistically significant differences in metabolic levels between wild-caught and domesticated shrimp using the t-test ( � for P < 0.05 and �� for P < 0.01). Daggers indicate statistically significant differences in metabolic levels between testes and vas deferens of wild-caught shrimp using the t-test ( † for P < 0.05 and † † for P < 0.01). Hashes indicate statistically significant differences in metabolic levels between testes and vas deferens of domesticated shrimp using the t-test (# for P < 0.05 and ## for P < 0.01). ND indicates that the designated metabolite was not detected in this analysis.

Quantitative analysis of eicosanoids and PUFAs in the testes and vas deferens of polychaete-and pellet-fed shrimp
To determine whether eicosanoid and PUFA profiles in the male reproductive tract were affected by shrimp diet, testes and vas deferens of polychaete-and pellet-fed shrimp were analyzed using UHPLC-HRMS/MS. First, levels of eicosanoids and PUFAs were compared between testes and vas deferens of shrimp in each feed group to determine metabolic changes during spermatogenesis and sperm maturation process, respectively. Data analysis revealed that the majority of the metabolites, including 15d-PGJ 2 (Fig 7C), (±)8-HETE (Fig 7D), 12(R)- Fatty acid profiles in polychaetes and feed pellets were analyzed using GC-FID. ND is abbreviated for not detected. Asterisks indicate significant differences between the average values of fatty acids found in polychaetes and feed pellets with the threshold for significance set at P < 0.05 ( � ) or P < 0.01 ( �� ).
https://doi.org/10.1371/journal.pone.0275134.t001 HETE (Fig 7F), (±)12-HEPE (Fig 7G), (±)18-HEPE (Fig 7H), ARA (Fig 7I), DHA (Fig 7J), and EPA (Fig 7K), were detected at higher levels in vas deferens than testes of shrimp in both feed groups, suggesting that these metabolites were more essential in the sperm maturation process than spermatogenesis. Meanwhile, levels of PGF 2α were comparable between testes and vas deferens of shrimp in both feed groups (Fig 7B). The two eicosanoids with distinct metabolic patterns according to feed types were PGE 2 and (±)11-HETE (Fig 7A and 7E). More specifically, levels of PGE 2 were comparable between testes and vas deferens of polychaete-fed shrimp (Fig 7A). In pellet-fed shrimp, however, PGE 2 was detected at similar levels in testes but became undetectable in vas deferens, suggesting that the use of feed pellets reduced the levels of PGE 2 in this organ. In contrast, (±)11-HETE was absent in most tested samples except in vas deferens of polychaete-fed shrimp (Fig 7E). These data suggest that although changes in shrimp diet did not alter relative levels of most PUFAs and eicosanoids in shrimp testes and vas deferens, the distribution of certain ARA-derived eicosanoids, namely PGE 2 and (±) 11-HETE, in vas deferens was affected by shrimp feed. Lastly, (±)8-HEPE and (±)15-HEPE were excluded from the analysis as they were quantifiable in less than 50% of samples. Results from other studies as well as data from our own analysis (Fig 6) revealed that polychaete-fed shrimp had higher total sperm counts than pellet-fed shrimp [18,20] However, the effects of shrimp diets on levels of PUFAs and eicosanoids in shrimp testes and vas deferens have yet to be investigated. In this study, levels of eicosanoids and PUFAs in testes were compared between polychaete-and pellet-fed shrimp to assess the impact of shrimp feed. Testes of polychaete-fed shrimp contained higher levels of (±)8-HETE (Fig 7D), but lower levels of 15d-PGJ 2 (Fig 7C), (±)12-HEPE (Fig 7G), (±)18-HEPE (Fig 7H), ARA (Fig 7I), DHA (Fig 7J), and EPA ( Fig 7K) than those in pellet-fed shrimp. On the other hand, levels of PGE 2 (Fig 7A), PGF 2α (Fig 7B), and 12(R)-HETE (Fig 7F) were comparable in testes of polychaete-and pelletfed shrimp. Lastly, (±)11-HETE (Fig 7E) was not detected in testes of shrimp from both feed groups, indicating that this compound was not involved in shrimp spermatogenesis.
A similar analysis was performed to compare levels of eicosanoids and PUFAs in vas deferens between polychaete-and pellet-fed shrimp. The UHPLC-HRMS/MS analysis revealed that PGE 2 ( Fig 7A) and (±)11-HETE (Fig 7E) were present only in vas deferens of polychaete-fed shrimp. As levels of these metabolites were below the limit of detection in vas deferens of pellet-fed shrimp, it is possible that the lack of these eicosanoids might be correlated with low sperm counts. On the other hand, levels of (±)12-HEPE (Fig 7G) were higher in vas deferens of pellet-fed shrimp than in those of polychaete-fed shrimp, suggesting a negative correlation between high levels of (±)12-HEPE and total sperm counts. Meanwhile, levels of PGF 2α (Fig  7B), 15d-PGJ 2 (Fig 7C), (±)8-HETE (Fig 7D), 12(R)-HETE (Fig 7F), (±)18-HEPE (Fig 7H), ARA (Fig 7I), DHA (Fig 7J), and EPA ( Fig 7K) were comparable in vas deferens of polychaete- . Error bars represent standard deviations. Asterisks indicate statistically significant differences in metabolic levels between polychaete-and pellet-fed shrimp using the t-test ( � for P < 0.05 and �� for P < 0.01). Daggers indicate statistically significant differences in metabolic levels between testes and vas deferens of polychaete-fed shrimp using the t-test ( † for P < 0.05 and † † for P < 0.01). Hashes indicate statistically significant differences in metabolic levels between testes and vas deferens of pellet-fed shrimp using the t-test (# for P < 0.05 and ## for P < 0.01). ND means that the designated metabolite was not detected. https://doi.org/10.1371/journal.pone.0275134.g007

PLOS ONE
and pellet-fed shrimp, indicating that the difference in shrimp feed did not affect the production of these eicosanoids in vas deferens.

Discussion
Poor reproductive performance in domesticated males is one of the contributing factors that delay the progress of shrimp aquaculture industry [29,30]. Although tremendous research efforts have been made to improve shrimp breeding, total sperm counts in domesticated males remain lower than those in wild-caught males [31,32]. In fact, studies have shown that the reproductive success of penaeid shrimp depends on various factors, including shrimp age, shrimp size, genetic background, rearing environment, hormones, and nutrients [20,[33][34][35][36]. As dietary PUFAs have been shown to improve sperm quality in crustaceans [17,37], it is likely that increasing PUFA consumption would affect levels of PUFAs and their downstream metabolites in the crustacean male reproductive tract. In this study, P. monodon testes and vas deferens were subjected to ethyl acetate and methanol-chloroform extraction, respectively. The organ extracts were then analyzed using UHPLC-HRMS/MS, revealing that a total of ten eicosanoids and three PUFAs were detected in shrimp testes and vas deferens. Correlations between metabolic profiles, organ types, and total sperm counts were then examined to assess the roles of PUFAs and eicosanoids in crustacean male reproduction.

Spermatophore quality between wild-caught and domesticated crustaceans
Spermatophore quality of decapod crustaceans can be evaluated using several parameters, including melanization, spermatophore weight, sperm number, sperm viability, sperm abnormality, and spermatophore absence rates [35,38]. The loss of spermatophore quality can be attributed to stress, poor nutrient, and the length of time spent in captivity for wild-caught shrimp [19,39]. In this study, all spermatophores were present and no melanization was observed in all collected samples. Wild-caught shrimp had higher spermatophore weights and higher total sperm counts than domesticated shrimp, suggesting that the spermatophore quality of wild-caught shrimp was higher than those of domesticated shrimp in this study. Our data were supported by Rodríguez et al. (2007), in which the wild-caught Pacific white shrimp Litopenaeus vannamei produced higher total sperm counts than the domesticated counterparts [32]. However, other studies reported that spermatophore weights and total sperm counts of wild-caught and domesticated shrimp were comparable [18,31]. The discrepancy between these studies may stem from the difference in shrimp size. A positive correlation between shrimp size and total sperm count has previously been reported in a different study in L. vannamei [40]. Upon closer examination of our data and the data from Rodríguez et al. (2007), it was confirmed that wild-caught males with higher body weights also had higher total sperm counts than domesticated males in both studies [32], whereas wild-caught and domesticated males with similar body weights also contained comparable total sperm counts [18,31]. As a result, shrimp body weight should also be taken into consideration during the comparison of total sperm counts between shrimp from different sources.

Correlations between levels of PUFAs in shrimp diets, shrimp male reproductive organs, and spermatogenesis
One of the contributing factors that affect sperm quality is the amounts of PUFAs in crustacean diets [41]. Supplementation of fish oil enriched in n-3 and n-6 PUFAs has been shown to increase levels of ARA, EPA, and DHA in P. monodon testis and enhance the number of spermatozoa in male broodstocks [41]. In fact, spermatophore quality can be used to determine the efficiency of crustacean maturation diets [18,42,43]. In this study, the consumption of polychaetes, which contained higher levels of n-3 and n-6 PUFAs than feed pellets, did not result in higher levels of ARA, EPA, and DHA in shrimp testis and vas deferens than those of pellet-fed shrimp. Moreover, a negative correlation between levels of dietary PUFAs and levels of PUFAs in testis and vas deferens was observed, suggesting that aside from the dietary intake, other factors also influenced levels of PUFAs in crustacean male reproductive organs.
In the oriental river prawn Macrobrachium nipponense, a positive correlation between high levels of n-6 PUFAs in the testis and crustacean spermatogenesis has been reported [44]. Levels of EPA and DHA in the testis increased as shrimp progressed from early to mid and late stages of gonad development [44]. Nevertheless, this observation might be species-specific as there was no correlation between levels of EPA and DHA in the testis and spermatogenesis or mating activities in M. rosenbergii [45]. On the other hand, high levels of ARA have typically been correlated with low sperm counts and poor sperm motility in mammals [46]. However, the effects of high levels of ARA in male reproductive organ have never been reported in crustaceans. In this study, the analysis of wild-caught and domesticated shrimp revealed a negative correlation between total sperm counts and high levels of EPA in testes as well as high levels of ARA, EPA, and DHA in vas deferens. These data were supported by the analysis of polychaeteand pellet-fed shrimp, in which higher levels of EPA were observed in testes of pellet-fed shrimp than those of polychaete-fed shrimp.

The identification of eicosanoids in the P. monodon male reproductive tract
As PUFAs are known precursors of eicosanoids, the increased levels of PUFAs in shrimp testis and vas deferens could potentially result in higher production of eicosanoids in these organs. In this study, the UHPLC-HRMS/MS analysis revealed that ten eicosanoids and three PUFAs were found in P. monodon testes and vas deferens. These included PGE 2 , PGF 2α , (±)8-HETE, (±) 11-HETE, 12(R)-HETE, (±)8-HEPE, (±)12-HEPE, and (±)18-HEPE, all of which had previously been identified in crustaceans [8, 9, 11-13, 15, 47-49]. Additionally, to the best of our knowledge, this is also the first identification of 15d-PGJ 2 and (±)15-HEPE in crustaceans. The roles of 15d-PGJ 2 in male reproductive maturation has been firmly established in mammals [4,50]. High levels of 15d-PGJ 2 in the testis and vas deferens were associated with impaired spermatogenesis in pigs and male infertility in humans, respectively [4,50]. In the testis, 15d-PGJ 2 acted through the reactive oxygen species (ROS) pathway, which prevented the differentiation of human testicular peritubular cells [4]. This resulted in the loss of contractility of the peritubular cells of the testis, which led to impaired spermatogenesis. On the other hand, high levels of 15d-PGJ 2 in vas deferens activated the PPARγ pathway, which regulated luminal electrolytes in the reproductive ducts that affected sperm functions and viability [50]. As high levels of 15d-PGJ 2 were detected in vas deferens of P. monodon, we propose that excess levels of 15d-PGJ 2 might impair sperm function and viability in shrimp vas deferens, which subsequently result in low sperm counts in penaeid shrimp.
Although the roles of 15d-PGJ 2 in spermatogenesis are well-established in mammals, the function of 15-HEPE in testis and vas deferens has not been reported in any organism. Nevertheless, the inhibition of 15-lipoxygenase, which converts EPA to 15-HEPE, can improve sperm motility and acrosome reaction rates as well as reduce the oxidative stress via ROS pathway [51]. Therefore, the identification of 15-HEPE in testis and vas deferens of domesticated shrimp might also indicate that the ROS pathway may be activated in domesticated shrimp.

Effects of eicosanoids in crustacean total sperm counts
In this study, the heat map analysis of relative abundance of PUFAs and eicosanoids in shrimp reproductive tract revealed that (±)8-HEPE and (±)8-HETE were the two most abundant eicosanoids in shrimp testes and vas deferens. In fact, high levels of (±)8-HETE and (±) 8-HEPE were reported in E. pacifica [52] and high levels of (±)8-HEPE were also detected in the hepatopancreas of P. monodon [49], suggesting that these hydroxy fatty acids were major metabolites and ubiquitously expressed in crustaceans.
To assess the roles of eicosanoids in shrimp male reproductive organs, two sets of shrimp samples were selected for analysis. Shrimp from different sources, namely wild-caught and domesticated shrimp, were used as representatives of shrimp with high and low total sperm counts, respectively. The effects of shrimp diets on total sperm counts were also examined as the use of polychaetes as live feed for male brooders has been shown to produce higher spermatophore weights and higher total sperm counts than the use of feed pellets [18,20]. The results from this study are summarized in Fig 8. The comparative analysis of levels of eicosanoids and PUFAs in testes and vas deferens revealed that levels of 15d-PGJ 2 , (±)8-HETE, and (±)12-HEPE in shrimp testes were lower than those in vas deferens in all shrimp samples ( Fig  8A), suggesting that these eicosanoids may be essential for the sperm maturation process.
Eicosanoid and PUFA profiles were also compared for shrimp from different sources (wildcaught vs. domesticated shrimp; Fig 8B) and for shrimp fed with different diets (polychaeteand pellet-fed shrimp; Fig 8C). In both sets of samples, high levels of (±)12-HEPE, (±) 18-HEPE, and EPA in testes as well as high levels of (±)12-HEPE in vas deferens were negatively correlated with total sperm counts (Fig 8B and 8C). In contrast, high levels of PGE 2 in vas deferens were positively correlated with high sperm counts in shrimp from both sets of samples. In humans, addition of PGE 2 and PGF 2α at low physiological levels to spermatozoa has been shown to improve sperm function [6]. Furthermore, transcriptomic analyses in crab gonads also provided supporting evidence regarding the positive effects of eicosanoid biosynthesis pathway in crustacean male reproductive maturation. This led to the identification of prostaglandin E synthase 2 and prostaglandin F synthase as candidates for the regulators of growth, sexual differentiation, and reproduction in the testis of the orange mud crab Scylla olivacea [53]. Similarly, prostaglandin E synthase and prostaglandin E2 receptor were also identified as potential regulators of gonadal development in P. trituberculatus [54]. These data were also supported by a study in mammals, in which cyclooxygenase-2 and prostaglandin synthase enzymes that regulate the conversion of ARA to PGE 2 could serve as a local modulator of testicular activity in Leydig and Sertoli cells [55]. Therefore, we propose that eicosanoids also serve as modulators for testicular development and sperm maturation process in P. monodon. Our results not only expand the coverage of eicosanoid biosynthesis pathway in crustaceans, but also suggest that the roles of eicosanoids in spermatogenesis are conserved between crustaceans and mammals. Furthermore, the correlations between total sperm counts and high levels of eicosanoids in shrimp testis and vas deferens also suggest an alternative approach to improve total sperm counts by increasing the prostaglandin biosynthesis while suppressing the production of HEPEs in the male reproductive tract of penaeid shrimp.
Supporting information S1 Table. Percentage of internal standards recovered from liquid-liquid extractions of P. monodon testes and vas deferens. Metabolic changes that occurred as sperm travels from testes to vas deferens in wild-caught and domesticated shrimp as well as in shrimp fed with different diets. (B) Metabolic changes in testes and vas deferens of wild-caught and domesticated shrimp, which represent shrimp with high and low total sperm counts, respectively. (C) Metabolic changes in testes and vas deferens of shrimp fed with polychaetes and feed pellets, which also resulted in high and low total sperm counts, respectively. Metabolites that share the same correlation in both sets of samples (shrimp source and shrimp feed) are underlined. https://doi.org/10.1371/journal.pone.0275134.g008